The characterization and preparation of various hormone and hormone-like receptors, including steroid, thyroid, and retinoid receptors such as those represented by the glucocorticoid, mineralocorticoid, thyroid, estrogen-related and retinoid classes has been subject of considerable research.
It is known, for example, that the glucocorticoid receptor belongs to a large superfamily of ligand-dependent transcription factors that have themselves diverse roles in homeostasis, growth and development. Comparison of complementary DNAs encoding these receptors, as well as mutational analyses of their coding sequences, have identified certain functional domains within the molecule that are thought responsible respectively for DNA binding, hormone binding and nuclear localization. See Evans, et al., Science 240, 889 (1988) for a review of this subject matter.
In the case of the glucocorticoid receptor, the so-called DNA binding domain spans some sixty-six amino acids and is highly conserved among various species. In addition, this domain has been found to be required in order to activate transcription. See Hollenberg, et al., Cell 49, 39 (1987), Miesfeld, et al., Science 236, 423 (1987), Danielsen, et al., Mol.Endo 1, 816 (1987), Kumar, et al., Cell 51, 941 (1987), Gronemeyer, EMBO J. 6, 3985 (1987), and Waterman, et al., Mol.Endo 2, 14 (1988). This domain has been found to contain nine invariant cysteine residues. Although the contribution of each cysteine residue to overall function is unknown, as is the actual structure formed by this domain, it has been proposed that these cysteine residues coordinate two zinc ions to form two DNA binding, so-called finger domains, which result in a ternary structure thought responsible for the localization and binding of the glucocorticoid receptor to the requisite DNA site. See Klug, et al., Tr.Biochem.Sci 12, 464 (1987), Bens, et al., Cell 52, 1 (1988), and Evans, supra.
In a location nearer the carboxyl-terminal end distal from the DNA binding region is the so-called ligand binding domain which has the demonstrated ability to block activity of the receptor in the absence of hormone. Thus, presence of the requisite hormone relieves the inhibition of the receptor to activity. Deletion of this region has been found to produce a hormone-independent transcription activator. See Godowski, et al., Nature 325, 365 (1987), Hollenberg, et al., supra, Kumar, et al., supra, Danielsen et al., supra, and Adler et al., Cell, 52, 685 (1988).
In contrast to these two domains, the sequences lying towards the amino-terminal region from the DNA binding domain are poorly understood both as to structure, and particularly, function. This region is extremely variable both in size and in composition amongst the various receptors--See Evans, supra--and may contribute to the heterogeneity of receptor function. See Kumar et al., supra, and Tora et al., 333, 185 (1988).
Despite extensive analysis, some of which has been reported in the scientific literature, the region(s) that determine(s) trans-activation of transcription initiation remains poorly characterized. Trans-activation domains can be defined as polypeptide regions that, when combined with the functional DNA binding domain, increase productive transcription initiation by RNA polymerases. See Sigler, Nature 333, 210 (1988), Brent et al., Cell 43, 729 (1985), Hope et al., Cell 46, 885 (1986), Ma et al., Cell 48, 847 (1987), Ma et al., Cell 51, 113 (1987), Lech et al., Cell 52, 179 (1988), and Hope et al., Nature 333, 635 (1988).
Previous research of the human glucocorticoid receptor by linker scanning mutagenesis identified two regions outside of the DNA binding region having a role in transcription activation. These regions were defined as T.sub.1 and T.sub.2. Giguere et al., Cell 46, 645 (1986). Further research from these laboratories has also resulted in the report of a co-localization of trans-activation and DNA binding functions. See Hollenberg et al., supra, Miesfeld, et al., supra, Danielsen et al., supra, and Waterman et al., supra. As a result, this research has given rise to an emerging picture of an increasingly modular molecule with discrete domains, each contributing to the identified properties of ligand-binding, DNA-binding and trans-activation of transcription. Until recently, the region(s) determining the trans-activation activity were not at all well understood. Thus, the picture based upon extant literature lacks an overall appreciation of the dynamic nature of the steroid receptors and how the various domains determine the cascade of events initiated by ligand-binding and consummated by promoter-specific trans-activation.
Further, although previous research has identified functional "domains", there has been little systematic effort to identify amino acids that contribute to the specific activities of the molecule itself. Thus, the previous identification of steroid receptor trans-activation regions resulted only from a demonstrated loss of activity via deletion or insertional mutagenesis, but in no case have the properties of the regions themselves been confirmed in assays that reflect a dominant gain of function. See also Ptashne, Nature 335, 683 (1988).
Thus, Godowski et al., Science 241, 812 (1988), report results that show that the glucocorticoid receptor contains at least one "enhancement domain" other than that overlapping the glucocorticoid response element binding region (i.e., the DNA binding domain) and that the second domain occupies a region near the receptor amino-terminus. Similarly, Webster et al., Cell 54, 199 (1988) report on an inducible transcription activation function of the estrogen and glucocorticoid receptors, and these researchers speculate that the relative positions of the hormone regions (i.e., ligand and DNA-binding domains) are not important for the transcription induction activity of the receptor. Yet, these researchers admit that they have no definition of the exact location and nature of what they call the hormone-inducible activating domain, to say nothing of its characterization and role in trans-activating potential.
The work by Giguere et al., supra, demonstrated a loss of activity in the glucocorticoid receptor based upon an assay measuring transcription activity, as a result of performing random site-mutagenesis at several locations of the molecule. As a follow-up, Hollenberg et al. deleted regions in the molecule, again demonstrating overall loss of transcription activity induced by such removal of stretches of amino acids.
The human glucocorticoid receptor (hGR) has served as a prototype, model receptor for gene regulation. As noted above, the DNA-binding and ligand-binding functional domains have been defined previously. Similarly, it has been found that these modular domains of the hGR receptor or other receptors may be moved to other parts of the receptor or attached to heterologous DNA-binding domains and still maintain function.
In contrast, relatively little is known about negative regulation by hGR. This is surprising in light of the key role that steroids play in development and negative feedback regulation. Glucocorticoid helps determine neural crest cell fate in the developing sympathoadrenal system, in part by repressing the induction of neural-specific genes [See Stein et al., Dev Bio 127, 316 (1988) and Anderson et al, Cell 47, 1079 (1986)]. Glucocorticoid also modulates the hypothalamic-pituitary-adrenal axis by inhibiting second messenger-induced peptide hormone induction. Recently, Akerblom et al. (Science 241, 350 (1988)) showed that the hGR negatively regulates the cyclic AMP-inducible alpha glycoprotein hormone promoter in asteroid and DNA-binding dependent manner. Wild-type expression is exhibited by a promoter of just 168 base pairs (termed alpha168). Basal expression in placental cells is mediated by factors bound to a 36 base pair palindromic cyclic AMP response element (CRE) cooperating with proteins binding to a 25 base pair tissue-specific element (TSE). Expression may be further enhanced through the CRE by the elevation of intracellular cyclic AMP levels. The hGR represses both the basal and cyclic AMP enhanced transcription in a glucocorticoid-dependent fashion. The transacting elements to which the hGR binds have been defined and are related to the consensus GRE sequence for activation. Similar research is reported by Sakai, et al., Genes and Development 2, 1144 (1988).